Student Theses and Dissertations

Thomas Hsiao

Date of Award

2018

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Steller Laboratory

Abstract

Apoptosis, which is one of many different types of programmed cell death, is widely utilized as a mechanism to eliminate unwanted cells. The ability of cells to undergo apoptosis is required not only for development in many organisms, but also to maintain proper homeostasis; too little apoptosis can result in cancer whereas too much can lead to degenerative diseases. Apoptosis has been well studied at events that take place at or downstream of the mitochondria; these steps are rigorous and predictable, making them easy to study. However, little is known about early, decision-making events that occur upstream of the mitochondria. It is important to understand these early decision-making steps because it is at these timepoints when cells are not yet committed to live or die, and therefore have great potential for therapeutic intervention. We have identified a novel ARTS-dependent complex that forms extremely early in apoptosis. After inducing mammalian cells to die, we fractionated the cell extracts using gel filtration and looked for changes in canonical apoptosis proteins. We observed that XIAP, the main caspase inhibitor in mammals, is recruited to a 5Mda complex as early as 30 minutes after stimulation of apoptosis. Furthermore, the recruitment of XIAP to the complex is dependent on the pro-apoptotic protein ARTS, as XIAP is not recruited to the complex when cells are knocked out for ARTS. Using mass spectrometry, we have identified the constituents of the complex. The identified subunits are Herc2, Neurl4, SSSCA1 and XIAP. None of the identified subunits, aside from XIAP, have been previously implicated in apoptosis. Using cell culture assays, we have shown that the novel complex acts to degrade XIAP, thus shifting the propensity of cells to undergo apoptosis early on in the decision-making process. We have shown that knocking down Neurl4 leads to the stabilization of XIAP during apoptosis and, consequently, reduces the number of cells that undergo apoptosis. The identification and elucidation of this novel complex in apoptosis not only increases our basic understanding of apoptosis, but also provides new therapeutic opportunities for modulating cell death.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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